Methods for the preparation of charged crosslinkers

ABSTRACT

Processes for the preparation of charged crosslinkers bearing a sulfonic acid moiety are disclosed. These procedures also optionally include methods to convert the resulting products to substantially a single salt form.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.14/539,361, filed Nov. 12, 2014, which is a continuation application ofU.S. Ser. No. 14/045,151, filed on Oct. 3, 2013, now issued as U.S. Pat.No. 8,921,566 on Dec. 30, 2014, which is a divisional application ofU.S. Ser. No. 13/315,005, filed on Dec. 8, 2011, issued as U.S. Pat. No.8,598,362 on Dec. 3, 2013, which claims the benefit of the filing dateunder 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/421,357,filed on Dec. 9, 2010. The entire content of each of theabove-referenced applications is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a process of preparing sulfonicacid-bearing crosslinkers. The sulfonic acid moiety can be a free acidor a salt or a mixture of free acid and salts. These linkers are used toprepare conjugates by covalently attaching two entities together, inwhich one entity contains an amino or a hydroxyl group and the otherentity contains a thiol group. The sulfonic acid moiety of these linkersimproves the aqueous solubility of the resulting conjugates.

BACKGROUND OF THE INVENTION

Compounds that can link together a thiol-bearing moiety and anamine-bearing moiety have found use in many diverse applications such asthe linkage of cytotoxic agents, fluorophores or metal chelating agentsto cell binding agents such as antibodies, growth factors or vitamins,or to large complexes such as liposomes (see U.S. Pat. Nos. 5,208,020;5,416,064; 5,846,545; 6,340,701; 6,716,821; 7,217,819; 7,276,497;7,388,026; 7,598,290 and U.S. Patent Publication No. 20100203007).Examples of such linkers include SPP, SPDP, SPDB and SMCC (FIG. 1) (seeU.S. Pat. Nos. 4,563,304; 6,913,748; 7,276,497; Widdison et al., 2006,J. Med. Chem., 49, 4392-4408; Phillips et al., 2008 Cancer Res., 68,9280-9290). Often one or both of the entities that are to be linkedtogether have only limited solubility in water which results in pooraqueous solubility of the resulting conjugate.

Recently, it has been shown that antibody-drug conjugates, wherein theantibody and the cytotoxic drug were linked via charged crosslinkers,especially those that comprise a sulfonic acid substituent showedseveral benefits over the corresponding conjugates prepared withnon-charged linkers (FIG. 2). These benefits include a) greater aqueoussolubility of the antibody-drug conjugate, b) ability to increase thedrug load on the antibody, while maintaining solubility and monomercontent and c) greater therapeutic activity, especially toward multidrugresistant cells (see U.S. Patent Publication No. 20090274713 &20100129314). Improved pre-clinical efficacy of antibody-drug conjugatesprepared with the sulfo-SPDB crosslinker [FIG. 2, compound (V)] thatcomprises a sulfonic acid substituent, has also been recently reported(Kovtun et al., 2010 European J. Cancer, Suppl, 8, p′76, Abstract #235).The only previously reported synthesis of sulfo-SPDB is a lengthy andinefficient process comprising of 6 chemical steps (FIG. 3, see U.S.Patent Publication No. 20090274713). Thus there is a need to provide asimplified and scalable process for the preparation of chargedcrosslinkers, such as sulfo-SPDB, that comprise a sulfonic acid group.These and other needs are addressed in the current invention.

SUMMARY OF THE INVENTION

The present invention describes processes for preparing sulfonicacid-bearing heterobifunctional linkers. These linkers bear athiol-reactive moiety to link one component, such as a cytotoxic drug,via a disulfide bond or a thioether bond, and an amine- orhydroxyl-reactive moiety to link a second component, such as a cellbinding agent, via an amide or ester bond. The sulfonic acid moiety ofthese linkers is located on the carbon alpha to the carbon bearing theamine or hydroxyl-reactive moiety and can exist as a free sulfonic acid,or as a salt. The invention also encompasses the conversion of thesulfonic acid in free acid or an undesired salt form to a substantiallyspecific desired salt form such as a sodium salt or a potassium salt.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of previously described non-charged linkers.

FIG. 2 shows the structures of sulfo-SPDB and an antibody-drug conjugateprepared with sulfo-SPDB.

FIG. 3 shows the synthesis of sulfo-SPDB from the prior art.

FIGS. 4A and 4B show the synthesis of sulfo-SPDB using the new method.

FIGS. 5A through 5E show the synthesis of disulfide-containingα-sulfonic acid crosslinking agents. J is H or a cation describedherein.

FIGS. 6A through 6D show the synthesis of maleimide-containingα-sulfonic acid crosslinking agents. J is H or a cation describedherein.

FIGS. 7A and 7B show the synthesis of haloacetamido-containingα-sulfonic acid crosslinking agents. J is H or a cation describedherein.

FIG. 8 shows the X-ray powder diffraction pattern of Crystalline Form 1of compound (V).

FIG. 9 shows the TGA and DSC profiles of Crystalline Form 1 of compound(V).

FIG. 10 shows GSV profile of Crystalline Form 1 of the compound (V).

FIG. 11 shows the X-ray powder diffraction pattern of Crystalline Form2a of compound (V).

FIG. 12 shows the TGA and DSC profiles of Crystalline Form 2a ofcompound (V).

FIG. 13 shows GSV profile of Crystalline Form 2a of the compound (V).

FIG. 14 shows the X-ray powder diffraction pattern of Crystalline Form 3of compound (V).

FIG. 15 shows the TGA and DSC profiles of Crystalline Form 3 of compound(V).

FIG. 16 shows GSV profile of Crystalline Form 3 of the compound (V).

FIG. 17 shows the X-ray powder diffraction pattern of Crystalline Form2b of compound (V).

DETAILED DESCRIPTION OF THE INVENTION

The invention discloses methods for the preparation of compounds thatcomprise a thiol reactive group, a sulfonic acid substituent and acarboxylic acid or a carboxylic acid derivative that is capable ofreacting with an amine or a hydroxyl group. Compounds that can beprepared by the process described herein are represented by formula (I)and (III):

or a salt thereof, wherein,

Q represents a thiol-reactive moiety. In one embodiment, Q is adisulfide group selected from, but not limited to, alkyl disulfide,phenyl disulfide, ortho or para-nitrophenyl disulfide, 2,4-dintrophenyldisulfide, pyridyl disulfide, nitropyridyl disulfide; a maleimido group,a haloacetyl group or haloacetamido group. Preferably, Q is selectedfrom a pyridyl disulfide, a nitropyridyl disulfide, a maleimido, abromoacetamido or iodoacetamido group. More preferably, Q is selectedfrom a pyridyl disulfide or a maleimido group. Still more preferably, Qis a pyridyl disulfide.

E represents a linear, branched or cyclic alkyl, alkenyl or alkynylhaving 1 to 10 carbon atoms, a phenyl group, a three to six memberedheterocycloalkyl group or a five or six-membered heteroaromatic group,containing 1, 2 or 3 heteroatoms selected from 0, N or S. Preferably, Eis selected from a linear, branched or cyclic alkyl containing 1 to 6carbon atoms, a phenyl or a piperidine group. More preferably, E is alinear alkyl having 1 to 4 carbon atoms. Still more preferably, E isrepresented by —CH₂CH₂—.

F is an optional moiety that has the same definition as E. Preferably, Fis a linear or branched alkyl having 1 to 4 carbon atoms. Still morepreferably, F is absent.

Alternatively, when G is absent, F and R₁ together with the carbon atomfrom which they are attached can form a three to seven memberedcycloalkyl group. Preferably, the cycloalkyl group is a cyclohexylgroup.

G is absent or represents -G′-(OCH₂CH₂)_(n) or -G′-(CH₂CH₂O)_(n),wherein n is 0 or an integer from 1 to 24; G′ is absent, —C(═O)NR₂— or—NR₂C(═O)—; and R₂ is H or a linear, branched or cyclic alkyl having 1to 10 carbon atoms. In one embodiment, G is —C(═O)NR₂—(CH₂CH₂O)_(n),wherein n is 0 or an integer from 1 to 5. In a preferred embodiment, R₂is H or Me. In another preferred embodiment, n is 2 or 4. Alternatively,G is an amino acid or a peptide unit, represented by -(AA)_(m)-, whereinAA is an amino acid residue; and m is 0 or an integer from 1 to 6. Inone embodiment, when m is 1, AA is preferably glycine or alanine. Inanother embodiment, m is 2 or 3. Preferably, G is gly-gly orgly-gly-gly. Even more preferably, G is absent.

R₁ is H or linear, branched or cyclic alkyl, alkenyl or alkynyl having 1to 10 carbon atoms. Preferably, R₁ is selected from H or a linear orbranched alkyl having 1 to 4 carbon atoms. More preferably, R₁ is H.

C(═O)L is a reactive ester or thioester group. In one embodiment, thereactive ester or thioester group is selected from but not limited to anN-hydroxysuccinimide ester, N-hydroxy sulfosuccinimide ester,nitrophenyl ester, tetrafluoro phenyl ester, pentafluorophenyl ester, athiopyridyl ester a thionitrophenyl ester. Preferably, the reactiveester group is an N-hydroxysuccinimide ester.

In one embodiment, for compounds of formula (I) and (III), G and F areboth absent, E is a linear or branched alkyl bearing 1 to 4 carbonatoms. Preferably, E is —CH₂—CH₂—.

In another embodiment, for compounds of formula (I) and (III), G isabsent and F and R₁ together forms a three to seven membered cycloalkylgroup. Preferably, the cycloalkyl group is cyclohexyl. More preferably,E is a linear or branched alkyl bearing 1 to 4 carbon atoms. Even morepreferably, E is —CH₂—.

One embodiment of the invention discloses a process for the preparationof compounds of formula (III):

or a salt thereof, comprising the following steps:

a) reacting a compound of formula (II)

or a salt thereof, with a sulfonating agent optionally in the presenceof a base to provide the compound of formula (III); wherein thedefinitions of Q, E, G, F and R₁ are as indicated above. Preferably, thesulfonating agent is selected from, but not limited to, chlorosulfonicacid and sulfur trioxide. More preferably, the sulfonating agent ischlorosulfonic acid. Preferably, the base is selected from, but notlimited to, trialkylamine, such as triethyl amine, diisopropylethylamine or tributyl amine, or 4-alkylmorpholine, such as4-methylmorpholine. More preferably, the base is diisopropylethyl amine;

b) Optionally purifying the product. Purification methods are known inthe art and include chromatography and/or crystallization. Suitableforms of chromatographic purification include but are not limited tosolid/liquid chromatography such as flash chromatography, mediumpressure chromatography, or high pressure liquid chromatography (HPLC),including normal phase, reverse phase, ion pair and ion exchangechromatography, super critical chromatography and forms of liquid/liquidchromatography such as counter current chromatography, dropletcountercurrent chromatography, centrifugal partition chromatography,high speed countercurrent chromatography, or a combination of the abovein any order. Preferably, the product is purified by reverse phasechromatography.

A second embodiment of the invention discloses a process for thepreparation of compounds of formula (I) or a salt thereof, comprisingthe following steps:

a) reacting a compound of formula (II)

or a salt thereof, with a sulfonating agent optionally in the presenceof a base to provide the compound of formula (III) or a salt thereof;wherein the definitions of Q, E, G, F and R₁ are as indicated above.Preferably, the sulfonating agent is selected from, but not limited to,chlorosulfonic acid and sulfur trioxide. More preferably, thesulfonating agent is chlorosulfonic acid. Preferably, the base isselected from, but not limited to, trialkylamine, such as triethylamine, diisopropylethyl amine (DIPEA) or tributyl amine, or4-alkylmorpholine, such as 4-methylmorpholine. More preferably, the baseis diisopropylethyl amine.

b) Optionally purifying the product. Purification methods are known inthe art and include chromatography and/or crystallization. Suitableforms of chromatographic purification include but are not limited tosolid/liquid chromatography such as flash chromatography or highpressure liquid chromatography (HPLC), including normal phase, reversephase, ion pair and ion exchange chromatography, super criticalchromatography and forms of liquid/liquid chromatography such as countercurrent chromatography, droplet countercurrent chromatography,centrifugal partition chromatography, high speed countercurrentchromatography, or a combination of the above in any order. Preferably,the product is purified by reverse phase chromatography.

c) Reacting the carboxylic acid moiety of the product of formula (III)or a salt thereof with a hydroxy or mercapto compound in the presence ofa coupling reagent to provide the compound of formula (I) or a saltthereof. Preferably, the hydroxy or mercapto compound is selected from,but not limited to, N-hydroxysuccinimide, N-hydroxy sulfo-succinimide,tetrafluorophenol, nitrophenol, dinitrophenol or thiopyridine. Morepreferably, the hydroxyl or mercapto compound is N-hydroxysuccinimide.Preferably, the coupling agent is selected from, but not limited toN,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ). More preferably, the coupling agent is EDC.

d) Optional purification of the resulting compound of formula (I) bychromatographic methods and/or crystallization by the methods listedabove.

The schematic representation of the process steps are below:

wherein, the definitions are as above; X is selected fromN-hydroxysuccinimide, N-hydroxy sulfo-succinimide, tetrafluorophenol,nitrophenol, dinitrophenol or thiopyridine; and J is H or a salt cationdescribed herein.

A third embodiment provides a process for the preparation of a compoundof formula (IV):

or a salt thereof, comprising:

a) reacting 4-(2-pyridyldithio)butanoic acid (2a) or a salt thereof witha sulfonating agent, optionally in the presence of a base to provide4-(2′-pyridyldithio)-2-sulfo-butanoic acid (IV) or a salt thereof.Preferably, the sulfonating agent is selected from chlorosulfonic acidor sulfur trioxide. More preferably, the sulfonating agent ischlorosulfonic acid. Preferably, the base is selected from, but notlimited to, triethyl amine, diisopropylethyl amine or tributyl amine.More preferably, the base is diisopropylethyl amine.

In one embodiment, the reaction of compound (2a) or a salt thereof witha sulfonating agent is carried out in the presence of1,2-di(pyridin-2-yl)disulfane (PySSPy). An amount of about 0.1 to about5.0 equivalent, preferably about 0.5 to about 1.0 equivalent of PySSPyrelative to compound (2a) can be used. Preferably, about 0.5 equivalent(e.g., 0.4, 0.5, 0.6 equivalent) of PySSPy can be used.

Alternatively, PySSPy can be added after the reaction or during theworkup of the reaction, such as during the extraction of the productfrom the reaction mixture.

4-(2-pyridyldithio)butanoic acid (2a) can be prepared according to knownprocedures in the art, such as those described in Widdison W C et al.Semisynthetic maytansine analogues for the targeted treatment of cancer.J Med Chem. 2006; 49:4392-4408, the entire teaching of which isincorporated by reference herein by its entirety.

b) Optional purification by chromatography and/or crystallization. Inone embodiment, the compound of formula (IV) is purified bychromatography. Suitable forms of chromatographic purification can bedetermined by one of ordinary skill in the art and include, but are notlimited to those described above. Preferably, the chromatography isreverse phase chromatography. In another preferred embodiment, thecompound of formula (IV) is purified on a silica column using a suitableeluting solvent or solvents. In a more preferred embodiment, thecompound of formula (IV) is purified on silica column using a mixture ofwater and one or more organic solvents as the eluting solvent. Forexample, the eluting solvent can be a mixture of water and acetonitrile,optionally with a small amount of an acid, e.g., acetic acid. In a morespecific embodiment, the eluting solvent is a mixture of water,acetonitrile and acetic acid in a volume ratio of 1:10:0.01.

A fourth embodiment provides a process for the preparation of a compoundof formula (V):

or a salt thereof, comprising:

a) reacting 4-(2-pyridyldithio)butanoic acid (2a) or a salt thereof witha sulfonating agent, optionally in the presence of a base to provide4-(2′-pyridyldithio)-2-sulfo-butanoic acid (IV) or a salt thereof.Preferably, the sulfonating agent is selected from chlorosulfonic acidor sulfur trioxide. More preferably, the sulfonating agent ischlorosulfonic acid. Preferably, the base is selected from, but notlimited to, triethyl amine, diisopropylethyl amine or tributyl amine.More preferably, the base is diisopropylethyl amine.

In one embodiment, the reaction of compound (2a) or a salt thereof witha sulfonating agent is carried out in the presence of1,2-di(pyridin-2-yl)disulfane (PySSPy). An amount of about 0.1 to about5.0 equivalent, preferably about 0.5 to about 1.0 equivalent of PySSPyrelative to compound (2a) can be used. Preferably, about 0.5 equivalent(e.g., 0.4, 0.5 or 0.6 equivalent) of PySSPy can be used.

Alternatively, PySSPy can be added after the reaction or during theworkup of the reaction, such as during the extraction of the productfrom the reaction mixture.

b) Optional purification by chromatography and/or crystallization. Inone embodiment, the compound of formula (IV) is purified bychromatography. Suitable forms of chromatographic purification can bedetermined by one of ordinary skill in the art and includes, but is notlimited to those described above. Preferably, the purification is byreverse phase chromatography. In another preferred embodiment, thecompound of formula (IV) is purified on a silica column using a suitableeluting solvent. In a more preferred embodiment, the compound of formula(IV) is purified on silica column using a mixture of water and organicsolvent as the eluting solvent. For example, the eluting solvent can bea mixture of water and acetonitrile, optionally with a small amount ofan acid, e.g., acetic acid. In a more specific embodiment, the elutingsolvent is a mixture of water, acetonitrile and acetic acid in a volumeratio of 1:10:0.01.

c) Reacting the carboxylic acid moiety of the compound of formula (IV)or a salt thereof with N-hydroxysuccinimide (NHS) in the presence of acoupling reagent to provide the compound of formula (V). Preferably, thecoupling agent is selected from, but not limited toN,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ). More preferably, the coupling agent is EDC.

In one embodiment, the reaction of compound of formula (IV) withN-hydroxysuccinimide is carried out in the presence of a base.Preferably, the base is selected from, but not limited to,trialkylamine, such as triethyl amine, diisopropylethyl amine ortributyl amine, or 4-alkylmorpholine, such as 4-methylmorpholine. Morepreferably, the base is diisopropylethyl amine.

d) Optional purification of the resulting compound of formula (V) bychromatographic methods and/or crystallization by the methods listedabove.

In one embodiment, the compound of formula (V) is purified bychromatographic methods described herein. In a particular embodiment,the compound of formula (V) is purified on a silica column using aneluting solvent. The eluting solvent optionally contains a small amountof a non-nucleophilic base, which includes, but is not limited to, atrialkylamine, such as triethyl amine, diisopropylethyl amine ortributyl amine, or 4-alkylmorpholine, such as 4-methylmorpholine.Preferably, the base is diisopropylethyl amine. In another embodiment,an ion exchange column can be used to further purify the compound offormula (V) following a purification on a silica column, particularlywhen a base is used in the reaction of compound of formula (IV) with NHSto form the compound of formula (V) or when a non-nucleophilic base isadded after the reaction, during the workup or during the purificationof compound (V). Suitable cation exchange columns are well known in theart, for example, those described in Sigma-Aldrich or Supelco catalogs.In one embodiment, the cation exchange resins are Amberlyst® 15 orDowex® 50WX4-200 resins.

In another embodiment, the compound of formula (V) is purified bycrystallization. In one embodiment, the crystallization is carried outby dissolving the compound of formula (V) in a hydrophilic solvent orsolvents, followed by placing the resulting solution at a reducedtemperature, for example, between about 0° C. to about 10° C., below 0°C., between about −10° C. to about 0° C., between about −20° C. to about−10° C., between about −30° C. to about −20° C., about −10° C., about−20° C. or about −30° C. Preferably, the reduced temperature is about−20° C. Any suitable hydrophilic solvent can be used. Exemplaryhydrophilic solvents include, but are not limited to, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile and a mixturethereof. Preferably, the hydrophilic solvent is acetone or acetonitrile.

In another embodiment, the compound of formula (V) can be purified bycrystallization in a mixture of organic solvent and water. Any suitableorganic solvents can be used. For example, the organic solvent can be awater miscible solvent, such as acetone, DMF, DMSO, acetonitrile or amixture thereof. In a preferred embodiment, crystallization solvent is amixture of acetone and water. More specifically, the ratio of acetoneand water is from about 80:20 v/v to about 99:1. Even more specifically,the volume ratio of acetone to water is 90:10 or 95:5. Crystallizationcan be carried out by dissolving the compound of formula (V) in thesolvent mixture, followed by placing the resulting solution at a reducedtemperature, for example, between about 0° C. to about 10° C., below 0°C., between about −10° C. to about 0° C., between about −20° C. to about−10° C., between about −30° C. to about −20° C., about −10° C., about−20° C. or about −30° C. Preferably, the reduced temperature is about−20° C.

In another embodiment, the compound of formula (V) can exist in a saltform, for example, when the reaction of compound of formula (IV) withN-hydroxysuccinimide is carried out in the presence of a base or when anon-nucleophilic base is added after the reaction, during the workup ofthe reaction or during the purification of compound (V). In oneembodiment, the compound of formula (V) is a triethylamine salt, adiisopropylethyl amine salt or a 4-methylmorpholine salt, preferably adiisopropylethylamine salt. The neutral form of the compound can beprepared by passing the salt through an ion exchange column as describedabove. Alternatively, the neutral form of the compound can be preparedby crystallization of the above-described salts in an organic solvent ora mixture of an organic solvent and water in the presence of a smallamount of an acid. Any suitable acid can be used. Exemplary acidsinclude, but are not limited to, trifluoroacetic acid (TFA), HCl, andH₂S0₄. Preferably, the acid is HCl or H₂SO₄. Any suitable amount of theacid can be used. For example, about 0.1 to 5 equivalent of the acid canbe used. Preferably, about 0.1 to about 1.5 equivalent of the acid isused. Even more preferably, about 0.5 to about 1.0 equivalent of theacid is used. Crystallization can be carried out in a suitable organicsolvent or organic solvents. Exemplary organic solvents include, but arenot limited to, acetone, DMF, DMSO and acetonitrile. Preferably, acetoneor acetonitrile are used for crystallization. Alternatively,crystallization can be carried out in a mixture of an organic solventand water. For example, acetone/water, DMF/water, DMSO/water andacetonitrile/water mixture can be used for crystallization.

In one embodiment, the invention is directed to new crystalline forms ofthe compound of formula (V), e.g., Crystalline Forms 1, 2a, 2b and 3 asdescribed below. Compound of formula (V) made according to the proceduredescribed in US 20090274713 is an amorphous solid and is highlyhygroscopic. Crystalline Forms 1, 2a, 2b and 3, particularly,crystalline Forms 1, 2a and 2b, are significantly less hygroscopic thanthe amorphous solid.

Crystalline Forms of Compound of Formula (V) Crystalline Form 1

In one embodiment, at least a particular percentage by weight of thecompound of formula (V) is the Crystalline Form 1 of the compound.Particular weight percentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, 100% or a percentage between 70% and 100%.

As used herein, “crystalline” refers to a solid having a highly regularchemical structure. When a particular percentage by weight of thecompound of formula (V) is a particular crystalline form, the remainderis some combination of amorphous form and/or one or more crystallineforms other than the particular form that is specified.

Crystalline Form 1 is characterized by the X-ray powder diffraction(XRPD) pattern shown in FIG. 8 with values of 2θ angle and relativeintensities as listed in Table 1, obtained using Cu Kα radiation. In aparticular embodiment, Form 1 is characterized by one, two, three, four,five, six, seven, eight or nine major XRPD peaks at 2θ angles of 4.2,12.6, 12.8, 13.8, 15, 20.1, 23, 25.5 and 37.5°. In another embodiment,Form 1 is characterized by major XRPD peaks at 2θ angles of 12.6, 12.8,15 and 23. It is to be understood that a specified 2θ angle means thespecified value±0.1°.

TABLE 1 Characteristic peaks of Form 1. 2θ angle (°) Intensity % 4.2 10012.6 31.1 12.8 22.1 13.8 10.2 15 20.8 16 10 18.7 29.4 18.9 24.4 19.415.9 20.1 12.2 21 13.6 22 15.9 23 19.1 23.6 12.9 24.5 11.9 25.5 14.625.7 17.2 27.5 12.2 27.8 9.5 29.1 9.8 29.4 14.3 37.5 11.6

As used herein, “major XRPD peak” refers to an XRPD peak with relativeintensity greater than 10%. Relative intensity is calculated as a ratioof the peak intensity of the peak of interest versus the peak intensityof the largest peak.

In one embodiment, Form 1 is characterized by a single endothermictransition at 99.7° C.±0.5° C. in the differential scanning calorimetry(DSC) profile shown in FIG. 9. The profile plots the heat flow as afunction of temperature. The DSC is performed on the sample using ascanning rate of 10° C./min from 25° C. to 240° C.

Crystalline Form 1 can also be characterized by the thermal gravimetricanalysis (TGA) profile shown in FIG. 9. The profile graphs the weightloss percentage of the sample as a function of temperature with thetemperature rate change being 10° C./min from ambient temperature to350° C. The profile shows a weight loss of approximately 3.7% as thetemperature of the sample changed from 40° C. to 200° C., whichindicates Form 1 is a monohydrate.

Form 1 is also characterized by gravitational vapor sorption (GVS)profile shown in FIG. 10. The profile shows the change in weight of asample as the relative humidity (RH) of the environment is changedbetween 0% and 90% at a 10% RH interval at 25° C. The adsorption profileshows a very small amount (1.6%) of weight gain between 40% RH and 90%RH. The desorption profile shows 1.9% weight loss between 90% RH and 0%RH, indicating no dehydration had occurred.

In one embodiment, Form 1 can be obtained by crystallization of thecompound of formula (V) in a mixture of an organic solvent and water.Suitable organic solvents are as described above. In a particularembodiment, a mixture of acetone and water is used for crystallization.The volume ratio of acetone and water can be in the range of 80:20 to99:1. Preferably, the ratio is 95:5. Crystallization can be carried outin a reduced temperature, preferably at −20° C.

Crystalline Form 2a

In one embodiment, at least a particular percentage by weight of thecompound of formula (V) is Crystalline Form 2a of the compound.Particular weight percentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, 100% or a percentage between 70% and 100%.

Crystalline Form 2a is characterized by the X-ray powder diffraction(XRPD) pattern shown in FIG. 11 with values of 2θ angle and relativeintensities as listed in Table 2, obtained using Cu Kα radiation. In aparticular embodiment, Form 2a is characterized by one, two, three, fouror five major XRPD peaks at 2θ angles of 8.6, 11.9, 16.5 and 24. Inanother particular embodiment, Form 2a is characterized by major XRPDpeaks at 2θ angles of 11.9, 16.4 and 24.

TABLE 2 Characteristic peaks of Form 2a 2θ angle (°) Intensity % 8.630.8 9 52 11.9 50.3 13.6 24.1 14.6 83.5 15.7 19.5 16.5 64.2 17.2 21.1 1826 18.6 34.3 18.9 33.5 19.8 100 20.9 30.3 21.3 65 22.4 47.8 23.2 59.1 2459.1 25.9 27.3 27.5 25.8 29.2 23.2 30.8 19.6

In one embodiment, Form 2a is characterized by a single endothermictransition at 201.9° C.±0.5° C. in the differential scanning calorimetry(DSC) profile shown in FIG. 121. Form 2a can also be characterized bythe thermal gravimetric analysis (TGA) profile shown in FIG. 12. Theprofile shows a weight loss of approximately 0.9% as the temperature ofthe sample changed from 40° C. to 90° C., which indicates Form 2a isanhydrous.

Form 2a is also characterized by gravitational vapor sorption (GVS)profile shown in FIG. 13. The adsorption profile shows a very smallamount (1.8%) of weight gain between 40% RH and 90% RH, indicating nohydration had occurred. The desorption profile shows 2.4% weight lossbetween 90% RH and O % RH.

In one embodiment, Form 2a can be prepared by crystallization ofcompound (V) in hydrophilic solvent or solvents as described above. In aparticular embodiment, Form 2a is prepared by crystallization in acetoneat a reduced temperature, such as −20° C.

In another embodiment, Form 2a can be prepared by removing the saltcation from a salt of compound (V), such as a DIPEA salt, by passing thesalt through a cation exchange column or by adding an acid. Subsequentcrystallization in a hydrophilic solvent, such as acetone oracetonitrile, gives Form 2a.

Crystalline Form 3

In one embodiment, at least a particular percentage by weight of thecompound of formula (V) is crystalline form 3 of the compound.Particular weight percentages include 70%, 72%, 75%, 77%, 80%, 82%, 85%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.9%, 100% or a percentage between 70% and 100%.

Crystalline Form 3 is characterized by the X-ray powder diffraction(XRPD) pattern shown in FIG. 14 with values of 2θ angle and relativeintensities as listed in Table 3, obtained using Cu Kα radiation. In aparticular embodiment, Form 3 is characterized by one, two, three, four,five, six, seven, eight, nine or ten major XRPD peaks at 2θ angles of10.4, 11.3, 12.2, 14.7, 17.6, 22.5, 26.3, 27.2, 28.8 and 29.8°. Inanother particular embodiment, Form 3 is characterized by major XRPDpeaks at 20 angles of 10.4, 1.3, 12.2, 17.6, 22.5, 26.3, 27.2 and 28.8°.

TABLE 3 Characteristic peaks of Form 3 2θ angle (°) Intensity % 9.3 30.810.4 23 11.3 19.6 12.2 16.8 14.6 61.9 14.7 24 16 19.8 17.6 59.6 17.958.7 18.4 66.1 21 100 21.9 29.8 22.5 32.6 23.5 24.1 24.5 16.4 24.6 26.926.3 27.1 27.2 54.6 27.6 18.2 28.8 35.4 29.8 25.3

In one embodiment, Form 3 is characterized by endothermic transitions at45.2±0.5° C., 93.3±0.5° C., and 177.2±0.5° C. in the differentialscanning calorimetry (DSC) profile shown in FIG. 15. Form 3 can also becharacterized by the thermal gravimetric analysis (TGA) profiled shownin FIG. 15. The profile shows a weight loss of approximately 4.48% asthe temperature of the sample changed from room temperature to 180° C.,which indicates Form 3 is a monohydrate.

Form 3 is also characterized by gravitational vapor sorption (GVS)profile shown in FIG. 16. The adsorption profile shows 25% of weightgain between 40% RH and 90% RH. The desorption profile shows 29.4%weight loss between 90% RH and O % RH. The second adsorption profileshows 0.4% weight gain between O % RH and 40% RH, indication dehydrationhad occurred during the desorption.

In one embodiment, Form 3 is prepared by crystallizing a salt (e.g.,DIPEA salt) in a mixture of an organic solvent and water containing asmall amount of an acid. Suitable organic solvents are as describedabove. In a particular embodiment, the organic solvent is acetone.Preferably, the volume ratio of acetone to water is about 80:20 to about99:1. Even more preferably, the ratio is about 90:10 to about 95:10. Anysuitable acids described herein can be used. In one embodiment, the acidis HCl or H2S04. An amount of about 0.1 to about 5 equivalent of theacid relative to the salt can be used. In a particular embodiment, about0.1 to about 1.5 equivalent of the acid, preferably about 0.5 to about1.0 equivalent of the acid can be used. Crystallization can be carriedout in a reduced temperature described herein, for example at about −20°C.

Crystalline Form 2b

In one embodiment, Crystalline Form 3 can be converted to an anhydrouscrystalline form, Crystalline Form 2b, by dehydration or drying undervacuum. In one embodiment, dehydration is carried out at 0% RH usingP₂O₅ as dessicant. In another embodiment, Crystalline Form 2b can beprepared by drying Crystalline Form 3 under vacuum at 30° C. for 24hours.

Crystalline Form 2b is characterized by the X-ray powder diffraction(XRPD) pattern shown in FIG. 17 with values of 2θ angle and relativeintensities as listed in Table 4, obtained using Cu Kα radiation. In aparticular embodiment, Form 2b is characterized by one, two, three, fouror five major XRPD peaks at 2θ angles of 8.5, 11.9, 16.4, 24 and 31.7 0.In another embodiment, Form 2b is characterized by a major XRPD peak at2θ angles of 31.7° and one, two, three or four major XRPD peaks at 2θangles of 8.5, 11.9, 16.4 and 24 0. In another particular embodiment,Form 2b is characterized by major XRPD peaks at 2θ angles of 11.9, 16.4,24 and 31.7°.

TABLE 4 Characteristic peaks of Form 2b 2θ angle (°) Intensity % 8.533.2 9.1 61.8 11.9 70 13.5 17.4 14.4 74.6 15.5 16.3 16.4 74.9 17.1 15.718.1 24 18.5 18.2 19.1 53.2 19.6 72.3 21.1 54.6 22.2 35.2 23.3 97.7 2460.3 25.8 21.4 27.6 36.6 29.3 21.4 30.6 13.8 31.7 100

A fifth embodiment of the invention discloses alternate methods ofpreparing compounds of the formula (III), wherein Q represents adisulfide moiety, or (IV). This embodiment provides a process for thepreparation of compounds of formula (3) or (IV) comprising:

a) reacting a lactone of formula (4) with a sulfonating agent to form acompound of formula (5) or a salt thereof. Preferably, the sulfonatingagent is chlorosulfonic acid or sulfur trioxide in its free or complexedform, such as a complex with dimethylformamide, or an amine exemplifiedby pyridine or triethyl amine. Preferably, the sulfonating agent issulfur trioxide in its free form.

b) reacting the compound of formula (5) or a salt thereof with asulfur-bearing nucleophile, optionally in the presence of a base and/oran acid or Lewis acid (Fujita et al., 1978 Tetrahedron Letters, 52,5211; Kelly et. Al. 1977, Tetrahedron Letters, 49, 3859). Preferably,the sulfur-containing nucleophile is selected from hydrogen sulfide, ora salt thereof, thiourea, thioacetic acid, 2-(trimethylsilyl)ethanethiolor thiophenol. More preferably, the sulfur-containing nucleophile isthiourea, thioacetic acid or thiophenol. Any suitable bases, acids orLewis acids known in the art can be used in the present methods.Preferably the base is selected from n-butyl lithium, lithiumdiisopropylamine, diisopropyl ethyl amine or triethyl amine. Morepreferably, the base is n-butyl lithium. The base, if used, in thesecases can be premixed with the thiol-bearing nucleophile then reactedwith a compound of formula (5). Alternatively, the base, if used, can beadded to a mixture of a compound of formula (5) and the thiol-bearingnucleophile. Preferably the base is premixed with the thiol-bearingnucleophile then reacted with a compound of formula (5). Preferably, theacid or Lewis acid, if used, is selected from 4-toluene sulfonic acid,aluminum trichloride, boron trifluoride, or titanium tetrachloride. Morepreferably, the acid or Lewis acid if used is 4-toluene sulfonic acid oraluminum trichloride. Typically such reactions are conducted at ambienttemperature in solvents such as dichloromethane, or 1,2-dichloroethanewhen a strong base is not employed. When a strong base is used reactionsare typically done at between −40 to 25° C.

c) Optional deprotection to release the free thiol. The deprotection canbe carried out by several means. Thioesters or thiouronium salts can behydrolyzed with bases such as NaOH or Na₂CO₃, (Zervas et. Al. 1963, J.Am. Chem. Soc. 85, 1337). Trimethylsilyl ethanol esters can also bedeprotected using fluoride containing agents such as hydrogen fluoride,or tetrabutylammonium fluoride (Hamm et. Al. 2004, Org. Lett. 6, 3817)and thiophenol adducts can be deprotected by electrolysis or bypalladium acetate (Chung et al, 2004, J. Chem. Soc. 126, 7386).Preferably, the deprotection involves hydrolysis with a base, such asNaOH or KOH. Typically such reactions are conducted in an aqueous NaOHor KOH solution, optionally mixed with a water-miscible solvent, such asmethanol, ethanol, THF, and at ambient temperature.

d) reacting the compound of formula (6) or a salt thereof with a mixeddisulfide compound to provide the compound of formula (3) or (IV).Preferably, the mixed disulfide compound is selected from2,2′-dithiodipyridine, 4,4′-dithiodipyridine,2,2′-dithiobis(5-pyridine), 4 nitrophenyldisulfide, S-methylmethanethiosulfonate or dimethyl disulfide. More preferably, the mixeddisulfide compound is 2,2′-dithiodipyridine. Other suitable disulfidecompounds are known in the art (see Aslam and Dent, 2000,Bioconjugation: Protein coupling techniques for the biomedical sciences,MacMillan, London)

Q′ is a linear, branched or cyclic alkyl having 1 to 10 carbon atoms,phenyl, ortho or para-nitrophenyl, dinitrophenyl, pyridyl ornitropyridyl; R_(a), R_(b), R_(c), R_(d) and R_(e), each independently,is H or a linear, branched or cyclic alkyl having 1 to 10 carbon atoms.Preferably, R_(a), R_(b), R_(c), R_(d) and R_(e), each independently, isH or a linear or branched alkyl having 1 to 4 carbon atoms. Morepreferably, R_(a), R_(b), R_(c), R_(d) and R_(e) are all H.

A more direct method of preparing (3) or (IV) would be the alphasulfonation of a thiolactone followed by hydrolysis of the resultingproduct and reaction with a disulfide forming agent.

A sixth embodiment provides a process for the preparation of compoundsof formula (3) or (IV) comprising:

a) reacting a thiolactone of formula (7) with a sulfonating agent toform a compound of formula (8) or a salt thereof; Preferably, thesulfonating agent is chlorosulfonic acid or sulfur trioxide in its freeor complexed form, such as a complex with dimethylformamide, or an amineexemplified by pyridine or triethyl amine. Preferably, the sulfonatingagent is sulfur trioxide in its free form;

b) hydrolyzing the compound of formula (8) or a salt thereof to form acompound of formula (6) or a salt thereof. Preferably, the hydrolysis iscarried out in the presence of NaOH or KOH.

c) reacting the compound of formula (6) or a salt thereof with adisulfide compound to provide the compound of formula (3) or a saltthereof. Preferably, the disulfide compound is selected from2,2′-dithiodipyridine, 4,4′-dithiodipyridine, 2,2′dithiobis(5-pyridine),4 nitrophenyldisulfide, S-methyl methanethiosulfonate or dimethyldisulfide. More preferably, the disulfide compound is2,2′-dithiodipyridine,

Q′ is a linear, branched or cyclic alkyl having 1 to 10 carbon atoms,phenyl, ortho or para-nitrophenyl, dinitrophenyl, pyridyl ornitropyridyl; R_(a), R_(b), R_(c), R_(d) and R_(e), each independently,is H or a linear, branched or cyclic alkyl having 1 to 10 carbon atoms.Preferably, R_(a), R_(b), R_(c), R_(d) and R_(e), each independently, isH or a linear or branched alkyl having 1 to 4 carbon atoms. Morepreferably, R_(a), R_(b), R_(c), R_(d) and R_(e) are all H.

In one embodiment, the compound of formula (3) prepared according theprocesses described in the fifth and sixth embodiments can react with ahydroxy or mercapto compound to form a compound of formula (Ia):

or a salt thereof, wherein R_(a), R_(b), R_(c), R_(d), R_(e), L and Q′are as described above.

Salts of the compounds of the present invention containing a carboxylicacid and/or sulfonic acid or other acidic functional group can beprepared by reacting the compounds with a suitable base. Suitable baseincludes, but is not limited to, alkali metal salts (especially sodiumand potassium), alkaline earth metal salts (especially calcium andmagnesium), aluminum salts and ammonium salts, tetraalkyl ammonium salts(such as tetramethyl ammonium, tetraethylammonium) as well as salts madefrom organic bases such as trimethylamine, triethylamine, and pyridine.Preferably, the salts are Nat or 1(t salts.

Particularly, the sulfonic acid moiety in the compounds described herein(e.g., compound of formula (I), (III) and (V)), can exist as a free acidor a salt thereof. Preferably a given batch of the compound would existsubstantially as a free acid or in a single salt form such as Na⁺, K⁺,NMe₄ ⁺ or the like. Such conversions can be conducted by several meansincluding but not limited to the following. A compound of formula (I),(III) or (V) can be brought into a solution or suspension with a bufferor buffer/solvent mixture containing the counter ion of interest inorder to exchange the ions. After which the desired compound or a saltbearing the cation of interest (J) can be purified from the mixture inseveral ways. Preferably, any water and solvent are evaporated and thedesired compound is dissolved in an organic solvent then filtered toremove any inorganic salts. Such filtration can be done through anordinary filter of through a relatively small amount of a filter aidsuch as but not limited to diatomaceous earth, silica, or alumina.Alternatively a solution of compound of formula (I), (III) or (V) iseluted through cation exchange material which has been conditioned withthe cation of interest. Alternatively a solution of the compound can bemixed in a “batch mode” with cation exchange material that has beenconditioned with the cation of interest to complete exchange then ionexchange material can be removed by filtration. Alternatively a solutionof the compound can be captured on anion exchange material then releasedby eluting in batch or chromatographic mode with solvent containing thedesired cation. Capturing by anion exchange chromatography followed bydisplacement with a cation of interest is most advantageous when thecation is volatile such as triethyl amine or diisopropyl ethyl amine.Examples of ion exchange materials which are appropriate for thisapplication are well known to one skilled in the art and include, Dowexresins, such as Dowex 1, Dowex 50, DEAE resins or Amberlite resins

The invention also encompasses methods for freezing a solution ofcompounds of formula (I), (III) or (V) and removing the frozen solventby lyophilization, also known as freeze drying. Solvents or mixtures ofsolvents that are suitable for this application should be able todissolve compounds of formula (I), (III) or (V) and the solvent orsolvents should be volatile under the vacuum conditions employed in thelyophilization process. The solvent or solvent system should also remainfrozen during the lyophilization process. Such solvents or solventsystems include but are not limited to water, 1,4-dioxane, tert-butanol,solutions of water and acetonitrile, solutions of water and methanol,solutions of 1-4-dioxate and tert-butanol. Each of these single solventsor mixtures of solvents can also contain an acidic or basic additivethat is volatile under the lyophilization conditions. Acidic or basicadditives that are suitable for this application include but are notlimited to formic acid, acetic acid, trifluoroacetic acid,triethylamine, or diisopropyl ethyl amine.

Schemes for the synthesis of compounds using the processes disclosed inthe present invention are shown in FIGS. 4-7. FIG. 4 shows the synthesisof N-succinimidyl 4-(2′-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB).4-(2-pyridyldithio)butanoic acid was prepared as previously described(Widdison et al., 2006, J. Med. Chem., 49, 4392-44080. Directsulfonation with chlorosulfonic acid in the presence of diisopropylethylamine (DIPEA) resulted in sulfonation at the C2 position to provide4-(2′-pyridyldithio)-2-sulfobutanoic acid. Reaction withN-hydroxysuccinimide (NHS) in the presence of EDC provided the desiredsulfo-SPDB crosslinker.

This direct sulfonation reaction can be utilized to produce a number ofα-sulfocarboxylic acids from carboxylic acids. Examples of the synthesisof a variety of such compounds from commercially available carboxylicacids are shown in FIGS. 5-7. These carboxylic acid compounds contain athiol reactive group, such a disulfide (FIG. 5), a maleimide (FIG. 6),haloacetyl or a haloacetamido (FIG. 7) to enable reaction with athiol-containing agent. The resulting sulfo-carboxylic acids can beconverted to active esters using a hydroxyl of mercapto compound in thepresence of a coupling agent, such as EDC, in an analogous manner tosulfo-SPDB shown in FIG. 4 and in the Examples to provide thebifunctional crosslinking agent. The active ester moiety can react witha cell binding agent or a cytotoxic compound or label bearing an aminoor hydroxyl group, while the disulfide, maleimide, haloacetyl orhaloacetamido moiety can undergo reaction with a cell binding agent or acytotoxic compound or a label bearing a thiol group, thus linking thecell binding agent with the cytotoxic compound or label.

DEFINITIONS

“Linear or branched alkyl” as used herein refers to a saturated linearor branched-chain monovalent hydrocarbon radical having one to twentycarbon atoms, preferably one to ten carbon atoms, more preferably one tofour carbon atoms. Examples of alkyl include, but are not limited to,methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl,—CH₂CH(CH₃)₂), 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl 3-pentyl,2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and thelike.

“Linear or branched alkenyl” refers to linear or branched-chainmonovalent hydrocarbon radical of two to twenty carbon atoms, preferablytwo to ten carbon atoms, more preferably two to four carbon atoms, withat least one site of unsaturation, i.e., a carbon-carbon, double bond,wherein the alkenyl radical includes radicals having “cis” and “trans”orientations, or alternatively, “E” and “Z” orientations. Examplesinclude, but are not limited to, ethylenyl or vinyl (—CH═CH₂), allyl(—CH₂CH═CH₂), and the like.

“Linear or branched alkynyl” refers to a linear or branched monovalenthydrocarbon radical of two to twenty carbon atoms, preferably two to tencarbon atoms, more preferably two to four carbon atoms, with at leastone site of unsaturation, i.e., a carbon-carbon, triple bond. Examplesinclude, but are not limited to, ethynyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like.

The terms “cyclic alkyl”, “cyclic alkenyl”, “cyclic alkynyl”, refer to amonovalent non-aromatic, saturated or partially unsaturated ring having3 to 10 carbon atoms as a monocyclic ring or 7 to 10 carbon atoms as abicyclic ring. Bicyclic carbocycles having 7 to 10 atoms can bearranged, for example, as a bicyclo [4,5], [5,5], [5,6] or [6,6] system,and bicyclic carbocycles having 9 or 10 ring atoms can be arranged as abicyclo [5,6] or [6,6] system, or as bridged systems such asbicyclo[2.2.1]heptane, bicyclo[2.2.2]octane and bicyclo[3.2.2]nonane.Examples of monocyclic carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-I-enyl,1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-I-enyl,1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl,cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and thelike.

The term “cycloalkyl” refers to a monovalent saturated monocyclic ringradical having 3 to 10 carbon atoms. Preferably, the cycloalkyl has 3 to7 carbon atoms in the ring. Examples of cycloalkyl include, not are notlimited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentyl, cyclohexyland cycloheptyl.

The term “alkyl” refers to linear, branched or cyclic alkyl describedabove.

The terms “heterocycloalkyl” are used interchangeably herein and referto a saturated or a partially unsaturated (i.e., having one or moredouble and/or triple bonds within the ring) carbocyclic radical of 3 to6 ring atoms in which at least one ring atom is a heteroatom selectedfrom nitrogen, oxygen, phosphorus, and sulfur, the remaining ring atomsbeing C, where one or more ring atoms is optionally substitutedindependently with one or more substituents described below. Preferably,heterocycloalkyl is fully saturated. A heterocycloalkyl may be amonocycle having 3 to 6 ring members (1 to 3 carbon atoms and 1 to 3heteroatoms selected from N, O, and S). Heterocycloalkyls areheterocycles described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566.

Examples of heterocycloalkyls include, but are not limited to,aziridine, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl,1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl,dihydrothienyl, dihydrofuranyl.

The term “heteroaromatic group” refers to a monovalent aromatic radicalof 5 to 12 membered monocyclic or bicyclic rings, containing one or moreheteroatoms independently selected from nitrogen, oxygen, and sulfur,wherein one or more ring atoms is optionally substituted independentlywith one or more substituents described below. Preferably, theheteroaromatic group is 5 or 6-membered monocyclic ring. Examples ofheteroaromatic groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl.

By way of example and not limitation, nitrogen bonded heterocycloalkylsor heteroaromatics are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or O-carboline.

The heteroatoms present in heteroaromatic groups or heterocycloalkylsinclude the oxidized forms such as NO, SO, and SO₂.

Unless otherwise indicated, the cyclic, branched or cyclic alkyl,alkenyl or alkynyl, cycloalkyl, heterocycloalkyl, phenyl, heteroaromaticgroup described herein can be optionally substituted. Suitablesubstituents include, but are not limited to, halogen, —OH, alkyl,alkoxy, haloalkyl, alkoxyalkyl, —NH₂, alkylamino, dialkylamino, whereinthe alkyl group in the substituent groups is unsubstituted linear orbranched alkyl having 1 to 4 carbon atoms. The heteroaromatic group andphenyl group can also be substituted with —CN and/or —NO₂ groups.

The term “alkoxy” refers to an alkyl radical attached through an oxygenlinking atom. “Alkoxy” can also be depicted as —O-alkyl. Preferably, thealkyl radical attached to the oxygen linking atom has 1 to 10 carbonatoms, preferably 1 to 4 carbon atoms. Examples of alkoxy include, butare not limited to, methoxy, ethoxy, propoxy, and butoxy.

The term “haloalkyl” refers to an alkyl radical substituted with one ormore (e.g., two, three, four, five or six) halogen groups. Preferably,the haloalkyl group has 1 to 10 carbon atoms, preferably 1 to 4 carbonatoms.

The term “alkylamino” refers to an alkyl radical attached through a —NHlinking group. “Alkylamino” can also be depicted as —NH-alkyl.Preferably, the alkylamino group has 1 to 10 carbon atoms, preferably 1to 4 carbon atoms.

The term “dialkylamino” refers to a group depicted as —N(alkyl)(alkyl).Preferably, each alkyl depicted in —N(alkyl)(alkyl) has 1 to 10 carbonatoms, preferably 1 to 4 carbon atoms.

The term “halo” or “halogen” refers to F, Cl, Br or I.

The term peptide is intended to include moieties comprising two or moresequentially linked amino acids selected from natural or unnatural aminoacids, including modified amino acids, such as, but not limited to,N-alkyl, N-aryl. Each of the said amino acids can be of theL-configuration, the D-configuration or racemic.

The term “amino acid residue” refers to an amino acid with the hydrogenatoms removed from the terminal carboxy and amino groups, i.e.,—NHCH(R)C(═O)O—, R is the side chain group.

The term “sulfonating agent” refers to an agent that can introduce asulfonic acid moiety in a compound. Sulfonating agents that may be usedin methods described herein are known in the art (see U.S. Pat. No.1,926,422; W. Thaler 1953, Macromolecules 16; 623-628; Truce and Olson,1953, J. Am. Chem. Soc., 75, 1651-1653) and include sulfur trioxide inits free or complexed form, such as complexed with dimethylformamide ordimethylacetamide, complexed with an amine, such as pyridine ortriethylamine. The sulfonating agent can also be a halosulfonic acid,such as chlorosulfonic acid and fluorosulfonic acid. Alkylsulfates, acidsulfites, sulfites and sulfuryl chloride may also be used (Kharasch andRead 1939, J. Am. Chem. Soc., 61, 3089-3092). There is also a reportsuggesting that lactones can undergo alpha-sulfonation with sulfurtrioxide (Patent DE 800410). The sulfonation reaction described hereincan be performed in solvents such as 1,4-dioxane, THF, ether orpoly-ether, acetonitrile, DMF, and halogenated solvents, Preferably thesulfonation reaction is performed in halogenated solvents such asdichloromethane, chloroform and 1,2-dichloroethane, more preferably in1,2-dichloroethane. Though sulfonation using chlorosulfonic acid ispreferred to be performed in a halogenated solvent, it can also beperformed in neat chlorosulfonic acid. Typically, the sulfonation isconducted at between 25−110° C. when chlorosulfonic acid is used as thesulfonating agent. Preferably, the sulfonation is conducted at between50−100° C., and more preferably between 70−90° C. When sulfur trioxideis used as the sulfonating agent reactions are typically conducted at−40 to 25° C., preferably −20 to 5° C.

The term “coupling reagent” refers to a reagent that activatescarboxylic acid group towards amide and ester formation. Couplingreagents that can be used in the processes described herein are known inthe art. Examples of coupling reagents include, but are not limited to,N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ). Typically these couplings are done at ambient temperature in anaprotic solvent, several suitable solvents that are typically employedare dichloromethane, tetrahydrofuran and dimethyl formamide. Couplingmethods and conditions are well known in the art (see Benoiton, 2006,Chemistry of Peptide Synthesis, CRC Press, Florida)

The term “a thiol reactive group” represents a group that can react witha thiol group in a thiol-containing compound. For example, a thiolreactive group can be a disulfide group or a maleimide, haloacetyl or ahaloacetamido, vinylsulfones, vinylsulfonamides, vinylpyridines,oxiranes or aziridines.

The term “amine or hydroxyl-reactive group” represents a reactive esteror thioester group that contains a leaving group that is readilydisplaced by an amine group or a hydroxyl group. Reactive ester orthioester groups are known in the art. For example, a reactive ester canbe an N-hydroxysuccinimide ester, N-hydroxy sulfosuccinimide ester,phthalimidyl ester, nitrophenyl ester, tetrafluoro phenyl ester,pentafluorophenyl ester, a thiopyridyl ester a thionitrophenyl ester.Other amine and hydroxyl-reactive groups are known in the art (see Aslamand Dent, 2000, Bioconjugation: Protein coupling techniques for thebiomedical sciences, MacMillan, London).

The term “sulfur-bearing nucleophile” refers to compounds bearing asulfur atom in which the sulfur atom can displace a leaving group orcause ring-opening on a separate compound. In the case of a lactone,sulfur nucleophile attack results in ring-opening to generate acarboxylic acid. Such reactions can be performed at room temperature orat elevated temperatures, such as 25 to 100° C. Such displacementreactions could occur in solution without additives or the displacementmay require the aid of an acid, Lewis acid or a base. Examples ofsuitable sulfur nucleophiles include hydrogen sulfide, sodiumhydrosulfide, thiourea and thioacetic acid. Other examples andappropriate reaction conditions are known to one skilled in the art (seeCremlyn, 1996, An introduction to organosulfur chemistry, Wiley, NewYork; Jerry March & Michael B. Smith, 2007 March's Advanced OrganicChemistry, Wiley, New York).

The term “salt” refers to a salt prepared from a compound of the presentinvention having an acidic functional group, such as a carboxylic acidor a sulfonic acid functional group, and an inorganic or organic base.Suitable bases include, but are not limited to, hydroxides of alkalimetals such as sodium, potassium, and lithium; hydroxides of alkalineearth metal such as calcium and magnesium; hydroxides of other metals,such as aluminum and zinc; ammonia, and organic amines, such asunsubstituted or hydroxy-substituted mono-, di-, or trialkyl amines;dicyclohexylamine; tributyl amine; triethyl amine, diisopropylethylamine (DIPEA) or tributyl amine, pyridine; 4-alkylmorpholine, such as4-methylmorpholine; N-methyl,N-ethylamine; diethylamine; triethylamine;mono-, bis-, or tris-(2-hydroxy-lower alkyl amines), such as mono-,bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, ortris-(hydroxymethyl)methylamine, N, N,-di-lower alkyl-N-(hydroxy loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine, ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like. The term “salt” also refers to a saltprepared from a compound of the present invention having a basicfunctional group, such as an amine functional group, and apharmaceutically acceptable inorganic or organic acid. Suitable acidsinclude, but are not limited to, hydrogen sulfate, citric acid, aceticacid, oxalic acid, hydrochloric acid (HCl), hydrogen bromide (HBr),hydrogen iodide (HI), nitric acid, hydrogen bisulfide, phosphoric acid,lactic acid, salicylic acid, tartaric acid, bitartratic acid, ascorbicacid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconicacid, glucaronic acid, formic acid, benzoic acid, glutamic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, andp-toluenesulfonic acid.

As used herein, C1S0₃H can be referred to as “chlorosulfonic acid,”“chloro sulfuric acid” or “sulfurochloridic acid” or other names thatone skilled in the art would readily recognize as representing C1S0₃H.

Unless otherwise indicated, bases, acids or Lewis acids that can be usedin the processes described above are any suitable bases known in theart. Examples of suitable bases include, but are not limited to,n-butyllithium, lithium diisopropylamine, diisopropylethylamine,triethylamine, tributylamine. Examples of suitable acids and Lewis acidsinclude, but are not limited to, benzosulfonic acid, 4-toluenesulfonicacid, sulfuric acid, hydrobromide, trifluoroacetic acid, aluminumtrichloride and titanium tetrachloride.

All patents, patent publications and non-patent literature cited hereinare expressly incorporated by reference in their entireties.

EXAMPLES Example 1 General Method for α-Sulfonation of Carboxylic Acids

To a stirred solution of the carboxylic acid compound acid in anhydrous1,2-dichloroethane (˜0.2 M) is added chlorosulfonic acid (5 equivalent)and the mixture is stirred in a 75° oil bath for 35 min. The heatingbath is removed and the mixture allowed to cool to ambient temperaturethen poured onto ice. The mixture is brought to pH 10˜11 by the additionof 10% NaOH and stirred for 10 min. Then the solution is adjusted to pH5 with 5% HCl and the mixture is transferred into a separatory funnel.The organic layer is extracted with deionized water and the aqueouslayers are combined, washed with dichloromethane and concentrated undervacuum until inorganic salts begin to appear on the wall of the flask.The remainder is diluted with CH₃CN and two layers formed aretransferred into a separatory funnel. The bottom light brownish layer isseparated, diluted with a small amount of deionized water, CH₃CN andformic acid (H₂O/CH₃CN/HCOOH, 10:1:1). It is loaded on a C18 column andpurified (eluting with CH₃CN/H₂O: 2% CH₃CN, 0-5 minutes; 2% to 28%CH₃CN, 5-20 minutes; 28-90% CH₃CN, 20-20:30 minutes; 90% CH₃CN,20:30-25:30 minutes; 90-2% CH₃CN, 25:30-26 min; 2% CH₃CN, 26-28 min) togive the desired sulfo-SPDB acid 3 as light brownish oil. The top CH₃CNlayer is concentrated under reduced pressure, diluted with H₂O and HCOOH(10:1), loaded on C18 column and eluted with CH₃CN/H₂O to give more ofthe sulfonated product acid.

Example 2

Preparation of 4-(2′-Pyridyldithio)-2-Sulfo-Butanoic Acid (IV) withoutAddition of Base in the Reaction

To a stirred solution of 4-(2-pyridyldithio)butanoic acid (2a) (804 mg,3.51 mmol) in anhydrous 1,2-dichloroethane (20 mL) was addedchlorosulfonic acid (1.4 mL, 21 mmol) quickly via a syringe. The mixturewas heated in a 75° C. oil bath and stirred at 73˜78° C. for 27 min. Theheating bath and the reaction mixture was allowed to cool to ambienttemperature and then the reaction mixture was poured onto ice. Themixture was brought to pH 10 with 10% NaOH and stirred for 10 min. Thesolution was adjusted to pH 5 with 5% HCl and the mixture wastransferred into a separatory funnel. The bottom organic layer wasseparated and extracted with deionized water. The aqueous layers werecombined, washed with dichloromethane and concentrated under vacuumuntil a slurry was obtained. The slurry was diluted with acetonitrile(100 mL) and a small amount of deionized water was added until all thesalts were dissolved and two layers formed. The layers were transferredto a separatory funnel. The bottom light brownish layer was isolated anddiluted with a deionized water 1 mL of the solution was taken, dilutedwith CH₃CN (0.1 mL) and formic acid (0.1 mL) and was loaded on a 250×21mm 10 micron C18 column. Elution with deionized water and acetonitrile(2% acetonitrile 0-5 min; linear gradient 2% acetonitrile-50%acetonitrile 5-23 min) gave the desired4-(2′-pyridyldithio)-2-sulfo-butanoic acid (IV) (R_(t)=11 min).Evaporation of solvent under vacuum gave 288 mg of desired product aslight brownish oil. The top CH₃CN layer was less clean than the lowerbrownish layer however additional product could be isolated from the toplayer as follows. Solvent was removed under vacuum, residue was taken upin CH₃CN, H₂O and HCOOH (10:10:1), and loaded on a 250×21 mm 10 micronC18 column. The column was eluted with CH₃CN/H₂O (2% CH₃CN, 0-5 minutes;linear gradient 2% to 28% CH₃CN, 5-20 minutes; 28-90% CH₃CN, 20-20:30min; 90% CH₃CN, 20:30-25:30 minutes; 90-2% CH₃CN, 25:30-26 minutes; 2%CH₃CN, 26-28 minutes) to give an additional 79 mg of (IV). ¹H NMR (400Hz, D₂O): δ 8.55 (d, J=6.0 Hz, 1H), 8.31 (t, J=8.0 Hz, 1H), 8.16 (d,J=8.4 Hz, 1H), 7.71 (t, J=7.2 Hz, 1H), 3.83 (dd, J₁=9.2 Hz, J₂=5.2 Hz,1H), 3.01-2.87 (m, 2H), 2.34-2.21 (m, 2H); ¹³C NMR (100 Hz, D₂O): 170.8,155.8, 145.6, 142.7, 125.8, 124.1, 64.0, 35.9, 27.3. MS (ESI) m/z 307.7(M-1).

Example 3 Alternative Synthesis of 4-(2′-Pyridyldithio)-2-Sulfo-ButanoicAcid (IV) with Addition of Base in the Reaction

4-(2-pyridyldithio)butanoic acid (2a) (102 mg, 0.45 mmol) wascoevaporated with 1,2-dichloroethane (2×6 ml), redisolved in 6 ml of1,2-dichloroethane and placed on preheated 75° C. oil bath.Chlorosulfonic acid (100 μL, 1.34 mmol) andN-ethyl-N-isopropylpropan-2-amine (117 μL, 0.67 mmol) were added and themixture was heated with stirring at 75° C. for 15 min. Another aliquotof chlorosulfonic acid (80 μl, 1.19 mmol) and DIPEA (63 μL, 0.36 mmol)was then added and the reaction was heated at 75° C. for an additional20 min. Analysis by HPLC indicated that the reaction was complete. Themixture was cooled in an ice bath and concentrated aqueous Na₂CO₃ addedto pH 11, stirred for 10 min, neutralized with H₃PO₄ to pH 7.0,concentrated, and purified on a C-18 column eluted with a gradient from100% of water (0.5% acetic acid) to 75% water (0.5% acetic acid)/25% ofMeOH to afford of 4-(2′-pyridyldithio)-2-sulfo-butanoic acid (IV) (67mg, 0.217 mmol, 48.7% yield). ¹H NMR (D₂O) 8.41 (dd, 1H, J=1.5, 4.9 Hz),7.91˜7.86 (m, 2H), 7.33˜7.30 (m, 1H), 3.75 (dd, 1H, J=5.1, 9.6),3.00˜2.94 (m, 1H), 2.86˜2.79 (m, 1H), 2.33˜2.26 (m, 2H); ¹³C NMR 176.60,160.28, 150.60, 140.27, 123.39, 122.92, 69.07, 37.56, 29.45; ESI MSm/z-307.8 (M-H).

Example 4 Preparation of N-Succinimidyl4-(2′-Pyridyldithio)-2-Sulfobutanoate (V) (Sulfo-SPDB)

4-(2′-pyridyldithio)-2-sulfo-butanoic acid (IV) (245 mg, 0.792 mmol),EDC-HCl (345 mg, 2.222 mmol) and 1-hydroxypyrrolidine-2,5-dione (120 mg,1.043 mmol) were stirred in DMA (8 mL) overnight and evaporated. Theproduct was purified by silica gel chromatography eluting with agradient of MeOH/CH₂Cl₂/HOAc (1:10:0.5% to 1:5:0.5%) to affordN-succinimidyl 4-(2′-pyridyldithio)-2-sulfobutanoate (V) (sulfo-SPDB).(258 mg, 0.635 mmol, 80% yield).

Example 5 Preparation of N-Succinimidyl4-(2′-Pyridyldithio)-2-Sulfobutanoate Sodium Salt (Vb) (Sulfo-SPDB NaSalt)

To a solution of N-succinimidyl 4-(2′-pyridyldithio)-2-sulfobutanoate(24 mg, 0.059 mmol) generated from silica gel column chromatography incold DMA (0.5 mL) was added aqueous NaH₂PO₄, (1.0 M, 2 mL, pH 5.5. Themixture was stirred on ice for 2˜3 min, evaporated under vacuum. Thesolid was suspended in 3 ml of DMA (3 mL), run through silica gel columneluted with 100% DMA. The fractions were pooled, evaporated and thencrystallized with MeOH/Et0H/toluene/hexane to afford the sodium salt ofN-succinimidyl 4-(2′-pyridyldithio)-2-sulfobutanoate (18 mg, 0.042 mmol,71.2% yield). ¹H NMR (DMF-d7) 8.49 (d, 1H, J=4.0 Hz), 7.88 (m, 2H), 7.27(m, 1H), 4.05 (dd, 1H, J=5.0, 9.4 Hz), 3.17˜3.08 (m, 2H), 2.92 (s, 4H),2.56 (m, 1H), 2.46 (m, 1H); ¹³C NMR 171.16, 166.61, 160.65, 150.66,138.81, 122.14, 120.37, 62.61, 36.63, 26.60; ESI MS m/z-404.7 (M-Na).

Example 6 Preparation of N-Succinimidyl4-(2′-Pyridyldithio)-2-Sulfobutanoate (V) (Sulfo-SPDB) 1. The Synthesisof 2-Sulfo-PBA

To a solution of 4-(pyridin-2-yldisulfanyl)butanoic acid (725 mg, 3.16mmol) and 1,2-di(pyridin-2-yl)disulfane (700 mg, 3.18 mmol) in 15 ml ofDCE (1,2-dichloroethane) at 75° C. was added chlorosulfuric acid (300μL, 4.51 mmol). After stirring for 20 min at 75° C., another portion ofchlorosulfuric acid (300 μL, 4.51 mmol) was added. The mixture wasallowed to stir at 75° C. for 20 min, then another portion ofchlorosulfuric acid (200 μL, 3.0 mmol) was added. Again 25 min later,the final portion of chlorosulfuric acid (200 μL, 3.0 mmol) was addedand the mixture was stirred for further 25 min. The mixture wasimmediately cooled on ice bath, neutralized with 1M NaOH to pH˜7,diluted with EtOAc/Hexane (1:1), separated, and the organic layer waswashed with pure water (3×25 ml) while the generated each of aqueouslayer was washed with EtOAc/Hexane (1:1, 35 ml). The aqueous layers werecombined, acidified with HCl/HOAc to pH 3˜4, concentrated to ˜10 ml,diluted with MeCN (100 ml), sonicated (or quickly stirred) for 1 h,filtered, and the resulting pellet washed with water/MeCN (1:10). Thesolution was then concentrated and purified on a Si0₂ cartridge (40 g)eluting with water/MeCN/HOAc (1:10:0.01). To the pooled fractionscontaining the product, DMF (˜5 ml) was added and evaporated to drynessto afford 4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid (295 mg, 0.954mmol, 30.2% yield).

2. The Synthesis of Sulfo-SPDB Linker

To a solution of 4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid (0.292mg, 0.944 μmol) in DMA (8 mL) was added 1-hydroxypyrrolidine-2,5-dione(0.120 mg, 1.043 μmol) andN1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine (EDC, 0.647mg, 4.17 mol). The reaction mixture was stirred overnight, evaporatedand purified on a Si0₂ cartridge (40 g) eluting with a mixture ofAcetone/DCM/HOAc (4:1:0.01). Product containing fractions were pooled,evaporated and solidified with EtOH/Tol/Hexane to afford1-(2,5-dioxopyrrolidin-1-yloxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonicacid (sulfo-SPDB, 0.270 mg, 0.664 mol, 70.4% yield).

Example 7

4-(pyridin-2-yldisulfanyl)butanoic acid (102 mg, 0.445 mmol) wascoevaporated with 1,2-dichloroethane, (2×6 ml) (to remove moisture) andre-dissolved in 6 ml of 1,2-dichloroethane. The resulting solution wasthen placed in a preheated oil bath (75˜80° C.). To this solution,sulfurochloridic acid (100 μL, ˜3 eq) was added and the resultingmixture was stirred for 5˜8 min. DIPEA (117 iμL, 1.5 eq) was then added(to precipitate the product) and the resulting mixture was stirred for 5min. Another portion of sulfurochloridic acid (40 ul, 1.33 eq) was addedand the mixture was stirred for ˜15 min, followed by addition of DIPEA(63 μL, 0.81 eq) and stirred for 5 min. A third portion ofsulfurochloridic acid (40 ul, 1.33 eq) was added and the mixture wasstirred for 15˜20 min. The resulting mixture was removed from the oilbath and cooled (on ice bath). Aqueous NaOH (0.5 M) or concentratedNa₂CO₃ was added to raise the pH˜12 and the reaction mixture was stirredfor 10 min. The organic phase was separated, washed with water (2×10ml), and neutralized with H₃PO₄ to pH 7. The organic phase was thenconcentrated to ˜5 ml and acidified with HCl to pH˜4. The crude productwas purified by passing through a C-18 column using eluting with agradient of 100% water (containing 0.5% HAc) to 75% of water (containing0.5% HAc)/25% of MeOH. Fractions containing the desired product (elutedout at 5˜10% of MeOH) were pooled, evaporated to afford4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid (67 mg, 0.217 mmol,48.7% yield). ¹H NMR (D₂O) 8.41 (dd, 1H, J=1.5, 4.9 Hz), 7.89 (m, 2H),7.31 (m, 1H), 3.75 (dd, 1H, J=5.1, 9.6), 2.97 (m, 1H), 2.82 (m, 1H),2.28 (m, 2H); ¹³C NMR 176.60, 160.28, 150.60, 140.27, 123.39, 122.92,69.07, 37.56, 29.45; ESI MS m/z-307.8 (M-H).

Alternative procedure: 4-(pyridin-2-yldisulfanyl)butanoic acid (106 mg,0.462 mmol) was coevaporated with 1,2-dichloreoethane (2×5 ml),redisolved in 5 ml of 1,2-dichloreoethane and placed in a preheated 75°C. oil bath. To this solution, sulfurochloridic acid (154 μL, 2.311mmol) and N-ethyl-N-isopropylpropan-2-amine (161 μL, 0.924 mmol) wereadded. The mixture was placed in the pre-heated 75° C. oil bath for 45min. Another portion of sulfurochloridic acid (45 μL, 0.675 mmol) wasadded and the reaction was heated at 75° C. for 1 more hour until thereaction was completed (monitored by HPLC). To the mixture was addedconcentrated Na₂CO₃ until pH 11. The resulting mixture was stirred for10 min, neutralized with H₃PO₄ to pH 7.5 and concentrated. The crudeproduct was purified by HPLC on a C-18 column eluting with a gradient of100% of water (0.5% HAc) to 80% water (0.5% HAc)/20% of MeOH to afford4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid (63 mg, 0.204 mmol,44.1% yield).

A mixture of 4-(pyridin-2-yldisulfanyl)-2-sulfobutanoic acid (245 mg,0.792 mmol),N1-((ethylimino)methylene)-N3,N3-dimethylpropane-1,3-diamine (345 mg,2.222 mmol) and 1-hydroxypyrrolidine-2,5-dione (120 mg, 1.043 mmol) werestirred in 8 ml of DMA overnight. The resulting solution was evaporatedand the residue was purified on a silica column using MeOH/CH₂Cl₂ (1:10to 1:5) containing 0.5% acetic acid as eluting solvent to afford1-(2,5-dioxopyrrolidin-1-yloxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonicacid (258 mg, 0.635 mmol, 80% yield).

To1-(2,5-dioxopyrrolidin-1-yloxy)-I-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonicacid (24 mg, 0.059 mmol) generated from SiO₂ column chromatography(eluted with 1:10:0.5% to 1:5:0.5% of MeOH/CH₂Cl_(2/)HAc) was added cold0.5 ml of DMA and 2 ml of 1.0 M NaH₂PO₄, pH 5.5. The mixture was stirredon an ice bath for 2˜3 min, evaporated using an oil pump (with no heat,free dry style). The resulting solid was suspended in 3 ml of DMA andpassed through a silica gel column eluting with 100% DMA. The fractionswere pooled, evaporated and then crystallized withMeOH/Et0H/Toluene/Hexane to afford sodium1-(2,5-dioxopyrrolidin-1-yloxy)-1-oxo-4-(pyridin-2-yldisulfanyl)butane-2-sulfonate(18 mg, 0.042 mmol, 71.2% yield). ¹H NMR (DMF-d7) 8.49 (d, 1H, J=4.0Hz), 7.88 (m, 2H), 7.27 (m, 1H), 4.05 (dd, 1H, J=5.0, 9.4 Hz), 3.17˜3.08(m, 2H), 2.92 (s, 4H), 2.56 (m, 1H), 2.46 (m, 1H); ¹³C NMR 171.16,166.61, 160.65, 150.66, 138.81, 122.14, 120.37, 62.61, 36.63, 26.60; ESIMS m/z-404.7 (M-Na);

Example 8 Instruments and Methods 1. X-Ray Powder Diffraction (XRPD)

X-Ray Powder Diffraction patterns were collected on a Bruker D8diffractometer using Cu Kα radiation (40 kV, 40 mA), θ-2θ goniometer,and divergence of V4 and receiving slits, a Ge monochromator and aLynxeye detector. The instrument is performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v2.5.0 and the data wereanalysed and presented using Diffrac Plus EVA v11.0.0.2 or v13.0.0.2.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. The sample was gently packed into a cavity cut intopolished, zero-background (510) silicon wafer. The sample was rotated inits own plane during analysis. The details of the data collection are:

-   -   Angular range: 2 to 42° 20    -   Step size: 0.05° 20    -   Collection time: 0.5 s/step

For Crystalline Form 3 the angular range was of 3 to 30° 20.

2. Differential Scanning Calorimetry (DSC)

DSC data were collected on a Mettler DSC 823e equipped with a 34position auto-sampler. The instrument was calibrated for energy andtemperature using certified indium. Typically 0.5-2.0 mg of each sample,in a pin-holed aluminium pan, was heated at 10° C./min from 25° C. to240° C. A nitrogen purge at 50 ml/min was maintained over the sample.The instrument control and data analysis software was STARe v9.20.

3. Thermo-Gravimetric Analysis (TGA)

TGA data were collected on a Mettler TGA/SDTA 851e equipped with a 34position auto-sampler. The instrument was temperature calibrated usingcertified indium. Typically 5-10 mg of each sample was loaded onto apre-weighed aluminium crucible and was heated at 10° C./min from ambienttemperature to 350° C. A nitrogen purge at 50 ml/min was maintained overthe sample. The instrument control and data analysis software was STARev9.20.

4. Gravimetric Vapour Sorption (GVS)

Sorption isotherms were obtained using a SMS DVS Intrinsic moisturesorption analyser, controlled by DVS Intrinsic Control softwarev1.0.0.30. The sample temperature was maintained at 25° C. by theinstrument controls. The humidity was controlled by mixing streams ofdry and wet nitrogen, with a total flow rate of 200 ml/min. The relativehumidity was measured by a calibrated Rotronic probe (dynamic range of1.0-100% RH), located near the sample. The weight change, (massrelaxation) of the sample as a function of % RH was constantly monitoredby the microbalance (accuracy±0.005 mg).

Typically 10 mg of sample was placed in a tared mesh stainless steelbasket under ambient conditions. The sample was loaded and unloaded at40% RH and 25° C. (typical room conditions). A moisture sorptionisotherm was performed as outlined below (2 scans giving 1 completecycle). The standard isotherm was performed at 25° C. at 10% RHintervals over a 0-90% RH range. Data analysis was undertaken inMicrosoft Excel using DVS Analysis Suite v6.0.0.7.

TABLE 5 Method Parameters for SMS DVS Intrinsic Experiments ParametersValues Adsorption - Scan 1 40-90 Desorption/Adsorption - Scan 2 90-0,0-40 Intervals (% RH) 10 Number of Scans 2 Flow rate (ml/min) 200Temperature (° C.) 25 Stability (° C./min) 0.2 Sorption Time (hours) 6hour time out

The sample was recovered after completion of the isotherm andre-analysed by XRPD.

5. Ion Chromatography (IC)

Data were collected on a Metrohm 861 Advanced Compact IC (for anions)using IC Net software v2.3. Accurately weighed samples were prepared asstock solutions in an appropriate dissolving solution and dilutedappropriately prior to testing. Quantification was achieved bycomparison with standard solutions of known concentration of the ionbeing analysed.

TABLE 6 IC Method Parameters for Anion Chromatography Type of methodAnion exchange Column Metrosep A Supp 5-250 (4.0 × 250 mm) ColumnTemperature (° C.) Ambient Injection (μl) 10 Detection Conductivitydetector Flow Raw (ml/min)  0.7 Eluent 3.2 mN sodium carbonate, 1.0 mMsodium hydrogen carbonate in 5% aqueous acetone.

Example 9 Crystallization Procedures for Crystalline Forms 1, 2a, 2b and3 1. Crystalline Form 1

531 mg of the DIPEA salt of sulfo-SPDB (sulfo-SPDB.0.6DIPEA) wasslurried in 1 ml of Acetone/H₂O (95/5 v/v) and magnetically stirred atabout −20° C. for 16 hours. The sample was filtered on a 0.45 μm WhatmanAutocup and washed with 2 ml of acetone. The sample was dried at roomtemperature for 6 hours and weighed. 113 mg of Crystalline Form 1 wascrystallized (Yield: 35%).

2. Crystalline Form 2a

a) Removing DIPEA from the Sulfo-SPDB DIPEA salt using cation exchangemedia.

1.09 g of Crystalline Form 1 was obtained from crystallization of 5.92 gof the DIPEA salt of sulfo-SPDB (sulfo-SPDB.0.6DIPEA) in 20 ml ofacetone at about −20° C. Crystalline Form 1 was filtered (Yield: 29%)and the liquor was carefully retrieved (liquor 1).

Liquor 1 was concentrated under reduced pressure to afford 4.84 g ofamorphous Sulfo-SPDB DIPEA salt. The salt was dissolved in 50 ml ofacetonitrile and eluted on 13.7 g of Amberlyst® 15 (Sigma-Aldrich,216380-dry, moisture ≦1.5%, 8 eq.) with about 220 ml of acetonitrile(liquor 2). The resin was initially washed and equilibrated inacetonitrile and water and then equilibrated with acetonitrile beforeuse.

Liquor 2 was concentrated under reduced pressure to give 1.37 g ofamorphous Sulfo-SPDB (Yield: 37%).

b) Crystallization procedure

Amorphous Sulfo-SPDB (1.37 g) was diluted in 10 ml of acetone, seededwith 21 mg of Crystalline Form 1 and placed under magnetic stirring at−20° C. for 3 hours. The sample was filtered on a 0.45 μm WhatmanAutocup and the vial was washed with 150 ml of acetone and 150 ml ofTBME. The sample was dried at room temperature for 16 hours and weighed.722 mg of Crystalline Form 2a was recovered (Yield: 64%).

3. Crystalline Form 3

79 mg of the supplied DIPEA salt of sulfo-SPDB (sulfo-SPDB.0.6DIPEA) wasslurried in 200 μI of Acetone/H₂0 (90/10 v/v). 110 μl of a stocksolution of HCl in Acetone/H₂0 (90/10) v/v (1.0 eq.) was added to theslurry at room temperature. The sample was observed to become a lightslurry and was placed under magnetic stirring for 16 hours at −20° C.The sample was filtered on a 0.45 μm Whatman Autocup and washed with 1ml of acetone. The sample was dried at room temperature for 6 hours andweighed. 43 mg of Crystalline Form 3 was recovered (Yield: 84%).

Example 10 Preparation of Crystalline Form 2b from Crystalline Form 3

Crystalline Form 2b was also obtained from Crystalline Form 3 bydehydration at 0% RH using P₂O₅ as desiccant or by drying under vacuumat 30° C. for 24 hours.

1-44. (canceled)
 45. A process for the preparation of a compoundrepresented by formula (V):

or a salt thereof, comprising the steps of: a) reacting a compound offormula (2a):

or a salt thereof, with a sulfonating agent to form a compound offormula (IV):

or a salt thereof; b) reacting the compound of formula (IV), or a saltthereof, with N-hydroxysuccinimide in the presence of a base to form asalt of the compound of formula (V); and c) purifying the sail of thecompound of formula (V) by crystallization in the presence of an acid toform the neutral form of the compound of formula (V).
 46. The process ofclaim 45, wherein the sulfonating agent is chlorosulfonic acid or sulfurtrioxide.
 47. The process of claim 45, wherein the sulfonating agent ischlorosulfonic acid.
 48. The process of claim 47, wherein thesulfonating reaction is carried out in the presence of PySSPy.
 49. Theprocess of claim 48, wherein about 0.5 equivalent of PySSPy is present.50. The process of claim 47, wherein the reaction between the compoundof formula (IV) and N-hydroxysuccinimide is carried out in the presenceof a coupling agent.
 51. The process of claim 50, wherein the couplingagent is selected from N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-diisopropylcarbodiimide (DIC) and 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ).
 52. The process of claim 51, wherein the coupling agent is EDC.53. (canceled)
 54. The process of claim 45, wherein the base isdiisopropylethylamine (DIPEA).
 55. The process of claim 45, wherein theprocess comprises the steps of: a) reacting the compound of formula (2a)or a salt thereof with chlorosulfonic acid to form the compound offormula (IV); and b) reacting the compound of formula (IV) withN-hydroxysuccinimide in the presence of EDC to form the compound offormula (V). 56-104. (canceled)
 105. The process of claim 45, whereinthe acid is HCl.
 106. The process of claim 55, wherein the acid is HCl.107. The process of claim 45, wherein the crystallization is carried outin a mixture of an organic solvent and water.
 108. The process of claim55, wherein the crystallization is carried out in a mixture of anorganic solvent and water.
 109. The process of claim 106, wherein thecrystallization is carried out in a mixture of an organic solvent andwater.